1,25(OH)2-vitamin D3 affects the subcellular distribution of protein kinase C isoenzymes in muscle cells.
Previous studies have shown the involvement of protein kinase C (PKC) in 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] regulation of DNA synthesis (long-term effect) and Ca2+ channel activity (short-term effect) in cultured myoblasts. Both events mediate stimulation of myoblast cell proliferation and growth by 1,25(OH)2D3. To characterise further the role of PKC in the hormone mode of action in muscle cells, the presence of PKC isoenzymes in chicken embryo myoblasts and changes in their total cell and subcellular levels after treatment (72 h and 5 min) with 1,25(OH)2D3 (1 nM), 12-O-tetradecanoyl phorbol 13-acetate (TPA; 100 nM) and 1,2-dioctanoyl-rac-glycerol (DOG; 50 microM) were investigated. Western blot analysis provided evidence on the expression of PKC alpha, beta and delta isoforms in avian myoblasts. Two immunoreactive bands of 80 kDa (intact molecule) and 50 kDa (catalytic fragment) were detected for each isoenzyme. 1,25(OH)2D3 and DOG, which increased myoblast PKC activity parallel with the stimulation of DNA synthesis and culture growth and the phorbol ester TPA which induced the opposite changes, exerted differential effects on PKC isoenzymes. Long-term (72 h) treatment with 1,25(OH)2D3 and DOG did not change total PKC isoform levels but decreased the 80 kDa species and increased the release of the catalytic fragment of PKC delta and beta, whereas TPA augmented the total amounts of the three PKC isoforms, increasing the band of 80 kDa of PKC beta and delta and the 50 kDa species for PKC alpha. Subcellular distribution studies showed that the 80 kDa molecule is only present in the cytosolic fraction whereas in the particulate fractions the 50 kDa fragments are detected. Increased amounts of the catalytic fragments of PKC beta and delta both in the nucleus and membranes were observed after 72 h treatment with DOG while 1,25(OH)2D3 increases PKC beta in the nucleus and PKC delta in membranes. TPA induced the appearance of the 50 kDa species of PKC alpha in the nuclear and membrane fractions. The phorbol ester also decreased the catalytic fragments of PKC beta and delta in membranes. Increased levels of PKC beta, and to a lesser extent of PKC delta, in membranes and cytosol could be detected after short exposure (5 min) of myoblasts to 1,25(OH)2D3, DOG and TPA. In conclusion, the data indicate the operation in myoblasts of PKC signal transduction pathways mediated by the Ca(2+)-dependent PKCs alpha and beta and the Ca(2+)-independent PKC delta. Moreover, the results suggest that the beta and delta isoforms of PKC could play a role in the regulation of muscle cell metabolism by 1,25(OH)2D3.
Marinissen
M J
MJ
Departamento de Biología, Bioquímica y Farmacia, Universidad NacionalDel Sur. San Juan, Bahía Blanca, Argentina.
Capiati
D
D
Boland
R
R
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002117:Calcitriol / Q000494:pharmacology*; D002478:Cells, Cultured; D002642:Chick Embryo; D004247:DNA / Q000096:biosynthesis; D004075:Diglycerides / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D009132:Muscles / Q000187:drug effects / Q000201:enzymology*; D011493:Protein Kinase C / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D004075:Diglycerides; D007527:Isoenzymes; C044295:1,2-dioctanoylglycerol; D004247:DNA; D011493:Protein Kinase C; D002117:Calcitriol; D013755:Tetradecanoylphorbol Acetate